(1) Background: Bacterial infections have long threatened global public safety; hence, it is significant to continuously develop antibacterial fibers that are closely related to people's daily lives. Berberine hydrochloride is a natural antibacterial agent that has application prospects in the preparation of antibacterial fibers. (2) Methods: This study firstly verified the antibacterial properties of berberine hydrochloride and its possible antibacterial mechanism. Thereafter, berberine hydrochloride was introduced into the self-made melt-spun polyurethane fiber through optimized coating technology. The performance of coating modified polyurethane fiber has been systematically evaluated, including its antibacterial properties, mechanical properties, and surface wettability. (3) Results: Results show that the antibacterial polyurethane fiber with desirable comprehensive properties is expected to be used in the biomedical fields. (4) Conclusions: The research also provides a reference for the development and application of other natural antibacterial ingredients in fiber fields.